NEW MODELS OF OCULAR INFLAMMATION AND NEW WAYS TO STUDY THEM: (1) Birdshot chorioretinopathy (BC) is strongly associated with HLA-A29. The mechanistic basis for the disease may be clarified by developing and studying HLA-A29 transgenic mice, but repeated attempts to derive a strain with a phenotype have not yet been fruitful. We now derived a new HLA-A29 construct. Founders are being observed for signs of pathology. (2) We continue to collaborate with the group of Dr. Warren Strober (NIAID) on ocular inflammation in NLRP3 knock-in mice. This gene is associated with Muckle-Wells syndrome, which among other pathologies is also associated with inflammation at the ocular surface. Studies with these mice indicate existence of an abnormal response to what we believe to be the normal flora of the ocular surface. These studies may provide new insights into ocular involvement in Muckle-Wells syndrome. (3) A new model of conjunctivitis has been developed by challenging the mucosal ocular surface with innate stimuli. Findings reveal that the conjunctiva-associated lymphoid tissue (CALT) responds dynamically and contains a variety of leukocytes, among them innate-like lymphocytes (ILCs) and gamma-delta T cells. We are defining the response and functionality of these cells in controlling infection at the ocular surface. We are also investigating whether there is a commensal ocular microbiome that functions to keep pathogenic organisms at bay. The insights may have important implications on treatment of ocular surface disease. FUNDAMENTAL MECHANISMS IN TOLERANCE, IMMUNITY AND AUTOIMMUNITY TO RETINAL ANTIGENS (1) Recent findings in IRBP spontaneously uveitic T cell receptor transgenic R161H mice indicated that commensal flora contributes to development of spontaneous uveitis. Mechanistic studies revealed that signaling through the clonotypic TCR in the gut by a non-cognate antigen derived from gut microflora is a necessary (though possibly not sufficient) stimulus (Horai et al, Immunity 2015). We are currently attempting to dissect which component(s) of the flora is(are) responsible and whether human flora will support development of disease.. (2) crossing R161H mice to IFN-g deficient or IL-17 deficient mice unexpectedly revealed a major role for IFN-g in the spontaneous disease. IFN-g-/- R161H mice, but not IL-17-/- R161H mice, had severely reduced spontaneous uveitis scores. These findings raise the possibility that IL-17 produced by R161H cells in the gut, as described above, is a marker of the pathogenic cells but not necessarily the pathogenic cytokine itself. (Bing et al., in preparation) (3) Vitamin (VitA) derivatives are necessary for functional activation of immune cells (published literature). We previously demonstrated the importance of Vitamin A (VitA) and its metabolite, retinoic acid, in ocular immune privilege. Using mice made VitA deficient (VAD), we are studying the role of VitA in regulation of autoimmunity to retina. We found that T cell effector function that was acquired before onset of VAD is maintained in the VAD host. These findings may have clinical implications in geographical regions where dietary VitA is limiting. (Horai, Zhou et al, in preparation) (4) IL-22 has been reported to have both pro-inflammatory and protective effects, depending on the tissue and the model. We used IL-22 and IL-22-receptor deficient mice and anti-IL-22 antibodies to examine effects of IL-22 modulation on EAU. Our data suggest that IL-22, produced by inflammatory cells and acting on neuronal and glial cells, has a local anti-inflammatory and neuroprotective role in the eye. Current studies are addressing the question which cells in the eye are a source of IL-22 (Mattapallil et al., in preparation). (6) In chronically inflamed eyes of R161H mice we identified structures resembling tertiary lymphoid tissue. These structures are functional germinal centers where immune cells may be activated for effector function and antibody production, affecting the course of disease. (J. Kielczewski, J. Immunol. 2016). (7) IL-17 is a pathogenic cytokine in autoimmune diseases, including EAU. Recent studies in our lab indicate that IL-17 regulates its own production and that of other lineage-specific cytokines in Th17 cells by feedback inhibition. The cellular and molecular mechanisms are being studied. (Chong et al., in preparation). EFFECTS OF INNATE IMMUNE RESPONSES ON AUTOIMMUNITY: The innate immune response directly affects immunopathogenic processes and also impacts on adaptive immunity. (1) We previously identified a population of NKT cells that produce IL-17 independently of IL-6 and IL-21 (NKT17). We find that IL-17 production in NKT cells and in some other populations of innate lymphoid cells, may be produced via a unique signaling pathway that bypasses STAT3. In vivo experiments suggest that this pathway may have a function in protecting from infections at the ocular surface. (St.Leger, Hansen et al, submitted). (2) Innate immunity receptors have a major role in controlling susceptibility to uveitis. Collaboration with the Rosenzweig lab at OHSU, Portland, OR and with the Lin lab at the Cleveland Clinic, Cleveland OH, we have revealed that the SYK/CARD9 signaling pathway and the Complement anaphylatoxin receptors C3aR and C5aR are required for development of EAU pathology. (Lee et al., J. Immunol. 29016 and Zhang et al. J Leukoc Biol 2016). THE OCULAR MICROBIOME AND MUCOSAL IMMUNE RESPONSES AT THE OCULAR SURFACE: Mucosal sites such as the intestine, oral cavity, nasopharynx, and female reproductive tract all have their associated commensal flora. The surface of the eye (conjunctiva) is also a mucosal site, but existence of a resident microbiome on the ocular surface is highly controversial. We used a mouse model of ocular surface disease to study whether commensal microbes are present in ocular mucosa and modulate immunity. We found that IL-17 is constitutively produced within the conjunctiva-associated lymphoid tissue (CALT) and recruits neutrophils to the ocular surface in the steady state and after a bacterial challenge. IL-17 sources in CALT include T cells, T cells and innate lymphoid cells (ILCs), in that order. Notably, a strain of Corynebacterium isolated from ocular tissue of mice, and known to also colonize the ocular surface of humans, induced the conjunctival T cells to secrete IL17, which in turn modified the local inflammatory signature and barrier function. Elimination of this putative commensal by antibiotic treatment, or its introduction into non-colonized mice, correlated inversely with severity of an experimental Candida albicans infection. Our results thus indicate that a relationship exists between commensals and immune cells at the ocular surface, which is critical for maintenance of homeostasis and host defense within the ocular mucosa (St. Leger et al., in preparation).